The EIF3H-HAX1 axis increases RAF-MEK-ERK signaling activity to promote colorectal cancer progression

Eukaryotic initiation translation factor 3 subunit h (EIF3H) plays critical roles in regulating translational initiation and predicts poor cancer prognosis, but the mechanism underlying EIF3H tumorigenesis remains to be further elucidated. Here, we report that EIF3H is overexpressed in colorectal cancer (CRC) and correlates with poor prognosis. Conditional Eif3h deletion suppresses colorectal tumorigenesis in AOM/DSS model. Mechanistically, EIF3H functions as a deubiquitinase for HAX1 and stabilizes HAX1 via antagonizing βTrCP-mediated ubiquitination, which enhances the interaction between RAF1, MEK1 and ERK1, thereby potentiating phosphorylation of ERK1/2. In addition, activation of Wnt/β-catenin signaling induces EIF3H expression. EIF3H/HAX1 axis promotes CRC tumorigenesis and metastasis in mouse orthotopic cancer model. Significantly, combined targeting Wnt and RAF1-ERK1/2 signaling synergistically inhibits tumor growth in EIF3H-high patient-derived xenografts. These results uncover the important roles of EIF3H in mediating CRC progression through regulating HAX1 and RAF1-ERK1/2 signaling. EIF3H represents a promising therapeutic target and prognostic marker in CRC.

The data demonstrating that HAX1 is a substrate of the SCFbetaTrCP ubiquitin ligase complex are preliminary.1.Several previous studies have demonstrated that HAX1 interacts with the F-box protein FBXO25 (doi: 10.1002/jcp.30044;doi: 10.1038/nm.3740;doi: 10.1074/jbc.RA120.014616).The authors should test the specificity of the HAX1-betaTrCP binding by immunoprecipitating a panel of at least 5-6 F-box proteins including FBXO25, betaTrCP1 and its paralog betaTrCP2 and test their interaction with endogenous HAX1. 2. The putative betaTrCP-binding domain (phosphodegron) in HAX1 is not canonical -it does not contain the glycine as in established betaTrCP substrates.More importantly, the sequence proposed as phosphodegron is not conserved in other mammals (mouse and rat) as the threonine residue is missing.3. Have the authors tested whether silencing of both betaTrCP1 and 2 results in HAX1 stabilization by CHX chase? 4. Does overexpression of betaTrCP-deltaF-box induces HAX1 accumulation? 5. Figure 3E: the effect of betaTrCP overexpression on HAX1 stability is not clear.
Overall, the data supporting the model that HAX1 works as a scaffold protein enhancing the interaction between RAF1, MEK1 and ERK1, and potentiating ERK1/2 phosphorylation and activation are not conclusive.For instance, in Figure 4B there is no correlation between the efficiency of HAX1 silencing and the effect on ERK1/2 phosphorylation.Although shHAX1 #1 is more efficient than shHAX1 #2 in knocking down HAX1 in DLD1 and HCT116 cells, it has less effect on the phosphorylation of ERK1/2.Reviewer #2 (Remarks to the Author): In the study by Jin et al., the authors show that EIF3H expression is increased in colorectal cancer.Using colitis-induced CRC mouse models, heterozygous deletion of Eif3h resulted in a reduced tumor load and size.Furthermore, KD of EIIF3H could reduce tumor proliferation and invasion/migration in CRC cell lines.On a mechanistic levels, the authors propose that EIF3H directly interacts with HAX1, affecting its protein stability via deubiquitination and counteracting its ubiquitination by ß-TRCP.They found that HAX1 enhances the interaction of RAF/MEK/ERK, resulting in increased phospho-ERK levels, and thereby mediates the effect of EIF3H.Finally, the authors show that combined Wnt/MEK inhibition effectively reduces PDX growth in EIF3H high tumors.The results are extensive and interesting, and the experiments are well designed.There are a few open questions that should be adressed in a revised version.

Major:
1.The authors describe that Eif3h flox/flox X Villin-CreERT are embryonic lethal and that this is the result of complete loss-of-function.However, no tamoxifen was added, so in principle, no recombination should have happened.To assess the leakiness of the system, I would recommend to compare Eif3h fl/wt X Villin-CreERT +/-TAM.The knockout itself seems to be mild as shown in Figure 1D, and I would suggest to use a second method (immunoblot) to quantify protein levels.2. If EIF3H directly regulates HAX1 levels, there should be a difference in HAX1 protein/transcript levels in EIF3H high/low tumors.The authors could re-analyse publicly available datasets to validate this correlation.In this regard, it is also interesting if the genetic status of the tumors affect EIF3H levels, as the authors suggest that high Wnt activity (for instance mediated by APC mutations) could result in higher EIF3H levels.3. Since EIF3H is part of the eIF3 complex, any interference/depletion would result in a global change of translation, thereby affecting many cellular processes.How these global effects compare to the specific effect on HAX1 is a key question.The authors could assess global protein abundances via MS after EIF3H KD to provide an insight into this question.4. The authors used CMYC as a readout for the Wnt pathway, I would recommend to use AXIN2, as CMYC can be influenced by activity of the RAS-MAPK pathway as well.Also NCB0846 is not a well recognized Wnt inhibitor.Using ICG-001 or tankyrase inhibitors would be better.Maybe the authors can add one set of experiments with one of the two inhibitors.
Minor: 1.In Figure 6J, the concentrations used for Trametinib seem to be extremely high for such a potent inhibitor.Did the authors confuse uM and nM? 2. In some sections, the language could be improved and spelling mistakes should be avoided.
Reviewer #3 (Remarks to the Author): The amplification of eukaryotic initiation translation factor 3 subunit h (EIF3H) has been found in various cancers.However, whether and how EIF3H contributes to tumorigenesis remains ambiguous.In this study, Jin et al. employed AOM/DSS induced colorectal cancer model to determine the role of EIF3H in cancer development since it was remarkably up-regulated in human CRC samples.They found that depleting EIF3H in intestinal epithelial cells significantly impaired colitis-induced colorectal tumorigenesis.Mechanistically, they further demonstrated that EIF3H functioned as a deubiquitinase to antagonist the ubiquitination of HAX1 by TrCP, and hence impaired the degradation of HAX1.Next, they found that HAX1 interacted with RAF1 and activated RAF/MEK/ERK signaling while active Wnt/βcatenin signaling promotes the expression of EIF3H in colorectal cancers, which constructs a signaling axis of Wnt/β-catenin-EIF3H-HAX1-RAF/MEK/ERK that is responsible for colorectal cancer development.To validate this finding and translate it to clinic cancer treatment, authors determined whether blocking both Wnt/β-catenin signaling and ERK signaling could effectively inhibit the growth of colorectal cancer with high EIF3H expression by using PDX model, and indeed they found this combination exhibited an excellent efficacy.Overall, this is an excellent study with compelling experimental data, which would have important implications in colorectal cancer treatment.Authors could address my following comments to improve their manuscript.
Major points: Although overall authors provided pretty strong data to support their conclusion, how HAX1 binds to and activates RAF1 is ambiguous.Does HAX1 association promote RAF1 homodimerization or heterodimerization with BRAF since RAF dimerization is critical for its activation as well as substrate phosphorylation?Does HAX1 also interact with the other two RAF isoforms, ARAF and BRAF since they have quite similar structures, particularly the N-lobe of kinase domain that mediates HAX1-RAF1 interaction?In addition, the NTA motif phosphorylation of RAF1 is a marker for RAF1 activation, does co-expression of HAX1 with RAF1 trigger this event?If authors can address these questions, this manuscript will be greatly improved.

Minor points:
1.There are pretty much English grammar errors in the whole manuscript.Please check the manuscript carefully and correct spelling/grammar errors.For example, line 104, Eif3hfl/oxwt should be Eif3hflox/wt.2. Images in Figure 1D are not clear.Authors should provide high-resolution images.3. Figure 3B, the anti-TrCP immunoblot for the last co-immunoprecipitation assay, it's better for authors to do this immunoblot with the same membrane for anti-HAX1 immunoblot.

Response to Reviewers
Reviewer #1 (Remarks to the Author): In this manuscript Jin and colleagues propose that an EIF3H-HAX1 axis promotes RAF-MEK-ERK signaling and colorectal cancer growth/metastasis.The authors show that EIF3H is overexpressed in human colorectal cancer and that EIF3H deletion in mice reduces colitisinduced colorectal tumorigenesis.They also show that EIF3H induces human CRC cell growth, migration and invasion.They also demonstrate that HAX1 is targeted for degradation by the SCFbetaTrCP ubiquitin ligase complex and that HAX1 expression results in increased interaction between RAF1, MEK1 and ERK1 thus leading to ERK1/2 phosphorylation and activation.Finally, it is shown that the EIF3H/HAX1 axis promotes CRC tumorigenesis and metastasis in a mouse orthotopic cancer model and that targeting both Wnt and RAF1-ERK1/2 signaling pathways synergistically decreases tumor growth in EIF3H-high patient-derived xenografts.
In my opinion the molecular mechanisms proposed in this study are not supported by the data presented.
The data demonstrating that HAX1 is a substrate of the SCFbetaTrCP ubiquitin ligase complex are preliminary.
The authors should test the specificity of the HAX1-betaTrCP binding by immunoprecipitating a panel of at least 5-6 F-box proteins including FBXO25, betaTrCP1 and its paralog betaTrCP2 and test their interaction with endogenous HAX1.

Response:
We thank the reviewer for the questions and suggestions.We have constructed 5 Fbox proteins' plasmids with Flag-tag or HA-tag including βTrCP1, βTrCP2, FBXO25, FBXO1 and SKP2.Then we transfected those plasmids into HEK293T cells and performed exogenous co-IP assay (Response Figure 1 A-E).Our results showed that FBXO25, βTrCP1 and its paralog βTrCP2 could interact with endogenous HAX1, while the other two F-box proteins, SKP2 and FBXO1, could not.This was consistent with our mass spectrometry results, as no unique peptide corresponding to SKP2 or FBXO1 was identified.Co-IP experiments also showed that HAX1 still associated with F-box deleted mutant βTrCP1 (a ubiquitination deficient mutant in which the substrate binding moiety is intact) (Response Figure 1F).
Response Figure 1.The interaction between exogenous F-box proteins and endogenous HAX1 was determined by co-IP assay.HEK293T cells were transfected with Flag-F-box protein or HA-FBXO25 plasmid.The cell lysates from HEK293T transfected with Flag-F-box protein plasmid were pulled down with anti-Flag M2 beads and immunoblotted with the indicated antibodies.Cell lysates from HEK293T transfected with HA-FBXO25 plasmid were immunoprecipitated with either control rabbit IgG or HA antibodies followed by immunoblotting with indicated antibodies.
2. The putative betaTrCP-binding domain (phosphodegron) in HAX1 is not canonical -it does not contain the glycine as in established betaTrCP substrates.More importantly, the sequence proposed as phosphodegron is not conserved in other mammals (mouse and rat) as the threonine residue is missing.

Response:
We thank the reviewer for the questions.βTrCP recognizes a DSGXXS motif or its variants (for example, DSG/DDG/EEG/SSGXXS/E/D motifs) in which the serine residues are phosphorylated by specific kinases to allow binding to βTrCP (PMID: 18500245).Here we identified one noncanonical βTrCP binding degron motif (231 DSEGRT 236) in HAX1, which meets the requirement of two positively charged/phosphorylated residues for binding βTrCP.It has been reported that some proteins have no-glycine degron motif that interacts with βTrCP for subsequent ubiquitination and degradation, such as WEE1, p100, CDC25A, MCL1 and TIPE2 (PMID: 15070733, 11994270, 14603323, 17387146, 32188758, 19797085).We think our HAX1 belongs to this category.
3. Have the authors tested whether silencing of both betaTrCP1 and 2 results in HAX1 stabilization by CHX chase?
Response: We thank the reviewer for the suggestion.We used siRNA to silence βTrCP1 and βTrCP2.The silencing efficiency was examined by western blot (βTrCP1) or qRT-PCR (βTrCP2).We observed that silencing of both βTrCP and βTrCP2 resulted in HAX1 stabilization in CRC cells by CHX chase assay (Response Figure 3, also added to Revised Supplementary Fig. 3d).
Response Figure 3. HAX1 turnover rate was analyzed by CHX assay in HCT116 cells transfected with siRNA targeting βTrCP or βTrCP2.

Does overexpression of betaTrCP-deltaF-box induces HAX1 accumulation?
Response: We thank the reviewer for the question.Yes, the expression of HAX1 were not affected by overexpression of delta F-box βTrCP in the HCT116 cells (Response Figure 4, also added to Revised Fig. 3f), as delta F-box βTrCP was a ubiquitination deficient mutant.Overall, the data supporting the model that HAX1 works as a scaffold protein enhancing the interaction between RAF1, MEK1 and ERK1, and potentiating ERK1/2 phosphorylation and activation are not conclusive.For instance, in Figure 4B there is no correlation between the efficiency of HAX1 silencing and the effect on ERK1/2 phosphorylation.Although shHAX1 #1 is more efficient than shHAX1 #2 in knocking down HAX1 in DLD1 and HCT116 cells, it has less effect on the phosphorylation of ERK1/2.

Response
Response: Thanks a lot for reviewer insightful questions and constructive suggestions.To add more evidence, we used one more HAX1 silencing sequence (totally 3 shRNA sequence) to reperformed western blot analysis in two CRC cell lines.Based on the new results, we think there is a correlation between ERK1/2 phosphorylation and HAX1 silencing efficiency (Response Figure 6A, also added to Revised Fig. 4b).More importantly, we also performed co-IP experiments while increasing HAX1 expression.The results showed that increasing HAX1 expression enhanced the RAF1 homodimerization and heterodimerization with BRAF (Response Figure 6B-C, also added to Revised Fig. 4g-h).These results suggest that HAX1 Major: 1.The authors describe that Eif3h flox/flox X Villin-CreERT are embryonic lethal and that this is the result of complete loss-of-function.However, no tamoxifen was added, so in principle, no recombination should have happened.To assess the leakiness of the system, I would recommend to compare Eif3h fl/wt X Villin-CreERT +/-TAM.The knockout itself seems to be mild as shown in Figure 1D, and I would suggest to use a second method (immunoblot) to quantify protein levels.
Response: Thanks a lot for the reviewer's constructive comments.The purpose of using this mouse model is to study if knockout or knockdown of EIF3H has any effect on colon tumorigenesis.We believe there is a partial leakiness of Villin CreERT , since the embryonic lethal happened even without tamoxifen injection.That's why we choose the partial knock out model.Two partial knock out strategy (①: Eif3h flox/wt VS Eif3h flox/wt Villin CreERT , ALL inject TAM; ②: Eif3h flox/wt Villin CreERT -TAM VS Eif3h flox/wt Villin CreERT +TAM) both make sense.We agree that strategy ② really can assess the leakiness of the system.It is warranted to be investigated in the future.The reasons why we choose "①" are that: (1) To achieve our purpose, strategy "①" will maximize Eif3h expression difference between wt and partial KO group since the leakiness happen (The partial knockout efficiency was confirmed in the following Response Figure 7A-C by IF, WB and IHC).( 2) To avoid TAM effect and reduce breeding times.The disadvantage of "①" strategy is that partial leakiness of Villin CreERT in other organ may have influence on mice development to further affect our conclusions.In fact, we checked Eif3h heterozygous knockout mice (Eif3h flox/wt Villin CreERT VS Eif3 flox/wt ) during the development.Our data showed the partial knockout mice have normal development with no overt phenotype in the gut, normal mating and breeding, compared to their Eif3h flox/wt littermates (Revised Supplementary Fig. 2b   and c).
We apologize that we did not present the knockout efficiency clearly.We relabeled the immunofluorescence (IF) data and showed that representative images of IF of colon tissues obtained from the indicated tamoxifen-induced mice (Response Figure7A, Revised Fig. 1d).
As suggested by the reviewer, we also performed immunoblot and immunohistochemistry staining assays to show that the protein level of Eif3h was indeed decreased in Eif3h heterozygous knockout mice (Response Figure7B and C, also added to Revised Supplementary Fig. 2d and 2e). 2. If EIF3H directly regulates HAX1 levels, there should be a difference in HAX1 protein/transcript levels in EIF3H high/low tumors.The authors could re-analyse publicly available datasets to validate this correlation.In this regard, it is also interesting if the genetic status of the tumors affect EIF3H levels, as the authors suggest that high Wnt activity (for instance mediated by APC mutations) could result in higher EIF3H levels.
Response: Thanks a lot for the reviewer's comments.We analyzed CPTAC colon cancer cohort and TCGA-CRC cohort, and found that the HAX1 positive correlated with EIF3H expression in CPTAC colon cancer cohort (https://cprosite.ccr.cancer.gov/)(Response Figure 8A-B, also added to Revised Supplementary Figure 4a).We also analyzed changes in mRNA levels of Eif3h from datasets of colon tumors isolated from Apc Min/+ and AOM-treated mice (GSE5204), and found that Eif3h mRNA levels were elevated in tumors of Apc Min/+ and AOM-induced mice compared with normal mice colon tissues (Response Figure 8C, Revised Supplementary Fig. 8a).In TCGA database, EIF3H expression was positively correlated with the mRNA levels of AXIN2 and MYC, which are downstream genes of Wnt pathway (Response Figure 8D, also added to Revised Supplementary Fig. 8b).
Response Figure 8. (A-B) Spearman rank correlation analysis of EIF3H and HAX1 protein expression in CRC tumors from TCGA-CRC cohort and CPTAC colon cancer cohort.(C) Using GEO2R of PubMed (http://www.ncbi.nlm.nih.gov/geo/geo2r/),we assessed changes in Eif3h mRNA levels in Gene Expression Omnibus datasets of colon tumors isolated from Apc Min/+ and AOM-treated mice (GSE5204).(D) Spearman rank correlation analysis of EIF3H and AXIN2, MYC mRNA level in CRC tumors from TCGA-CRC cohort.
3. Since EIF3H is part of the eIF3 complex, any interference/depletion would result in a global change of translation, thereby affecting many cellular processes.How these global effects compare to the specific effect on HAX1 is a key question.The authors could assess global protein abundances via MS after EIF3H KD to provide an insight into this question.
Response: Thanks for the reviewer's question and suggestion.First, we measured the nascent protein synthesis in HCT116 with/without induction of EIF3H KD using the Click-iT® Plus OPP Alexa Fluor® 488 Protein Synthesis Assay Kit (PMID: 26319900).EIF3H KD resulted in partial inhibition of nascent protein synthesis compared to the control group (Response Figure .9A).As a positive control, the nascent protein synthesis is significantly decreased in Cycloheximide (CHX) treated group.Previous works have reported that overexpression of EIF3H in NIH-3T3 cells results in significant activation of protein synthesis, especially some oncogenic mRNAs involved in growth control (PMID: 17170115, 18544531, 23716667).We also measured the specific effect of EIF3H knockdown on the translational efficiencies of HAX1, CCND1 and ACTB mRNAs by polysome profiling assay.Although a shift of ribosomes toward the lightest polysomes, relative to the control cell, was observed in the EIF3H knockdown HCT116 cell (Response Figure 9C, also added to Revised Supplementary Fig. 4f), EIF3H knockdown could not reduce the polysome size of HAX1 mRNAs, as evidence by no shift of the polysome peak to smaller polysomes (Response Figure 9D, also added to Revised Supplementary Fig. 4g-h).This observation of HAX1 was similar to GAPDH mRNA reported by Zhang et.al (PMID: 17170115, PMID: 18544531).ACTB and CCND1 act as a negative and positive control (PMID: 18544531).Taken together, these results suggested that EIF3H knockdown has no effect on HAX1 protein synthesis, although EIF3H affects global nascent protein synthesis.We have added the results to Revised Supplementary Figure 4f-h, and the method to supplemental materials and methods.1.In Figure 6J, the concentrations used for Trametinib seem to be extremely high for such a potent inhibitor.Did the authors confuse uM and nM?
Response: We thank the reviewer to point out this mistake.In Figure 6J, the concentrations used for Trametinib was nM.We have corrected this mistake.
2. In some sections, the language could be improved and spelling mistakes should be avoided.
Response: Thank a lot for reviewer's kind reminder.We apologize for spelling and grammatical mistakes and have corrected them throughout the text accordingly.
Reviewer #3 (Remarks to the Author): The amplification of eukaryotic initiation translation factor 3 subunit h (EIF3H) has been found in various cancers.However, whether and how EIF3H contributes to tumorigenesis remains ambiguous.In this study, Jin et al. employed AOM/DSS induced colorectal cancer model to determine the role of EIF3H in cancer development since it was remarkably up-regulated in human CRC samples.They found that depleting EIF3H in intestinal epithelial cells significantly impaired colitis-induced colorectal tumorigenesis.Mechanistically, they further demonstrated that EIF3H functioned as a deubiquitinase to antagonist the ubiquitination of HAX1 by TrCP, and hence impaired the degradation of HAX1.Next, they found that HAX1 interacted with RAF1 and activated RAF/MEK/ERK signaling while active Wnt/β-catenin signaling promotes the expression of EIF3H in colorectal cancers, which constructs a signaling axis of Wnt/βcatenin-EIF3H-HAX1-RAF/MEK/ERK that is responsible for colorectal cancer development.
To validate this finding and translate it to clinic cancer treatment, authors determined whether blocking both Wnt/β-catenin signaling and ERK signaling could effectively inhibit the growth of colorectal cancer with high EIF3H expression by using PDX model, and indeed they found this combination exhibited an excellent efficacy.Overall, this is an excellent study with compelling experimental data, which would have important implications in colorectal cancer treatment.Authors could address my following comments to improve their manuscript.

Major points:
Although overall authors provided pretty strong data to support their conclusion, how HAX1 binds to and activates RAF1 is ambiguous.Does HAX1 association promote RAF1 homodimerization or heterodimerization with BRAF since RAF dimerization is critical for its activation as well as substrate phosphorylation?Does HAX1 also interact with the other two RAF isoforms, ARAF and BRAF since they have quite similar structures, particularly the N-lobe of kinase domain that mediates HAX1-RAF1 interaction?In addition, the NTA motif phosphorylation of RAF1 is a marker for RAF1 activation, does co-expression of HAX1 with RAF1 trigger this event?If authors can address these questions, this manuscript will be greatly improved.
Response: Thanks a lot for the reviewer's constructive suggestions.To test whether HAX1 promotes RAF1 homodimerization or heterodimerization with BRAF, we performed co-IP (pull-down) experiments using different tagged proteins.Cells were co-transfected with Myc-RAF1/Flag-BRAF or Flag-RAF1/Myc-RAF1 in the presence of increasing HA-tagged HAX1, then co-IP experiments were performed.Increasing amounts of HA-tagged HAX1 promotes the binding of Myc-RAF1/Flag-BRAF or Flag-RAF1/myc-RAF1.Thus, our results demonstrate that increasing HAX1 enhanced the RAF1 homodimerization and RAF1/BRAF heterodimerization (Response Figure 12 A-B, also added to Revised Fig. 4g-h).Similarly, when HAX1 was knockdown, the RAF1 homodimerization and heterodimerization with BRAF was decreased, but these phenomena can be rescued by HAX1 re-expression l (Response Figure 12C-D, also added to Revised Fig. 4i and Supplementary Fig. 7b).
Co-IP assay confirmed the interaction between HAX1 and the other two RAF isoforms: ARAF and BRAF (Response Figure 12E, also added to Revised Fig. 4i and Supplementary Fig. 7c).
Although ERK phosphorylation has been altered by HAX1 KD or overexpression accordingly, there was no change in NTA motif phosphorylation level of RAF1 (Phospho-Ser338) (Response Figure 12F-G, Revised Fig. 4b).Further studies are warranted for this observation.
Figure 4. Representative immunoblots showing HAX1 steady-state expression in HCT116 cells upon delta F-box βTrCP overexpression.5. Figure 3E: the effect of betaTrCP overexpression on HAX1 stability is not clear.Response: Thanks for the concern.We re-performed this CHX chase assay and replaced the less clear image for revised version (Response Figure 5, also added to Revised Fig. 3e).As showed in this figure, βTrCP overexpression led to accelerated turnover rate of HAX1, while the F-box mutant βTrCP had no effect.Response Figure 5. HCT116 cells transfected with either WT Flag-βTrCP or F-box mutant βTrCP were treated with CHX for the indicated times.The cell lysates were immunoblotted with indicated antibodies (left).The turnover rate of HAX1 was shown (right).
could promote ERK1/2 phosporylation level by enhancing the RAF1 homodimerization and heterodimerization with BRAF.Response Figure 6.(A) Effect of HAX1 knockdown on the phosphorylation of ERK1/2 in DLD1 and HCT116 cells was detected by western blotting.(B-C) The HEK293T cells were transfected with Myc-RAF1, Flag-BRAF (B) or Flag-RAF1 (C) with increasing HA-HAX1 plasmids.Cell lysates were immunoprecipitated with anti-Myc beads and immunoblotted with indicated antibodies.WCL, whole cell lysis.Reviewer #2 (Remarks to the Author): In the study by Jin et al., the authors show that EIF3H expression is increased in colorectal cancer.Using colitis-induced CRC mouse models, heterozygous deletion of Eif3h resulted in a reduced tumor load and size.Furthermore, KD of EIIF3H could reduce tumor proliferation and invasion/migration in CRC cell lines.On a mechanistic levels, the authors propose that EIF3H directly interacts with HAX1, affecting its protein stability via deubiquitination and counteracting its ubiquitination by ß-TRCP.They found that HAX1 enhances the interaction of RAF/MEK/ERK, resulting in increased phospho-ERK levels, and thereby mediates the effect of EIF3H.Finally, the authors show that combined Wnt/MEK inhibition effectively reduces PDX growth in EIF3H high tumors.The results are extensive and interesting, and the experiments are well designed.There are a few open questions that should be adressed in a revised version.

Response
Figure 7. (A) Representative images of IF staining of colon tissues obtained from the indicated tamoxifen-induced mice.Red arrows indicated the cells (expressing CRE-ERT and having recombination happen) that had less expression of Eif3h (purple) in Eif3h -/wt mouse when compared with Eif3h flox/wt mice.Nuclei stained with DAPI (blue).Scale bar = 20µm.(B) Protein levels of EIF3H in indicated mouse colon tissues.(C) Representative images of Immunohistochemistry staining of colon tissues obtained from the indicated tamoxifen-induced mice.Scale bar = 25µm.
Response Figure 9. (A-B) Evaluation of EIF3H KD on nascent protein synthesis.Two days after doxycycline induction, O-propargyl-puromycin (OPP) was added to the culture medium to label nascent peptides which were visualized by Immunofluorescence microscope after fixation with fluorescent Click iT chemistry.The fluorescence intensity of all the cells corresponding to protein synthesis.Representative images of protein synthesis were shown (A).Data represented means ± SD from three independent experiments (B).(C) Polysome profiles of EIF3H KD and control cells.HCT116 Cells were knocked down of EIF3H, further lysed and subjected to sucrose gradient centrifugation.The sucrose gradient profiles were obtained by continuous scanning at A254.The positions in the gradients of 40S subunits, 80S ribosomes, and polysomes were labeled.The translational status of indicated mRNA in EIF3H KD and control cells were examined by qPCR (D).EIF3H knockdown had no effect on HAX1 protein synthesis.4. The authors used CMYC as a readout for the Wnt pathway, I would recommend to use AXIN2, as CMYC can be influenced by activity of the RAS-MAPK pathway as well.Also NCB0846 is not a well recognized Wnt inhibitor.Using ICG-001 or tankyrase inhibitors would be better.Maybe the authors can add one set of experiments with one of the two inhibitors.Response: Thanks a lot for constructive suggestions.As suggested by the reviewer, we used ICG-001 in different concentration to treat CRC cells and checked the change of EIF3H mRNA/protein level.Treatment with ICG-001 reduced the expression of EIF3H at both mRNA and protein levels in a dose-dependent manner (Response Figure 10A-B).We also used AXIN2 as a readout for the Wnt pathway to re-analyze the mRNA samples from DLD1, HCT116 and RKO cells treated with Wnt pathway inhibitor NCB0846.The results showed that both EIF3H and AXIN2 transcription could be inhibited by NCB0846 (Response Figure 11A-B).All those results were added to Revised Fig. 6b-c, and Revised Supplementary Fig. 8c.Response Figure 10.qRT-PCR (A) and western-blot (B) analysis of EIF3H and AXIN2 level in DLD1 and HCT16 cells treated with different concentrations of Wnt pathway inhibitor ICG-001.Response Figure 11.qRT-PCR (A) and western-blot (B) analysis of EIF3H and AXIN2 levels in DLD1, HCT16 and RKO cells treated with different concentrations of Wnt pathway inhibitor NCB0846.Both EIF3H and AXIN2 transcription could be inhibited by NCB0846.Minor: